Reversible global ischemia leading to cardiac contractile dysfunction remains a significant problem in cardiac surgery. In the past 3 years, we have investigated potential mechanisms responsible for this dysfunction including altered calcium regulation, loss of high energy phosphates and oxygen free radical injury. We have determined that the energetic state of the heart is only mildly depressed post-ischemia and does not limit contractile recovery. In fact, we have developed methods for significantly improving ATP preservation during ischemia by promoting anaerobic glycolysis for energy generation and preventing cytosolic calcium accumulation during ischemia. This preservation solution significantly prolongs tolerance to ischemia in normal adult, neonatal and hypertrophied myocardium. Contractile dysfunction however, particularly after prolonged ischemia, remains a clinically relevant problem. We have concluded that the mechanism(s) likely responsible for contractile dysfunction post- ischemia are altered calcium regulation for activation/contraction coupling, and/or the response (sensitivity) of contractile proteins to calcium. We have developed a method of measuring cytosolic calcium from the whole heart using a visible light fluorescent probe, Rhod2, and will directly determine the role cytosolic calcium regulation and contractile proteins play in the post-ischemic heart. Since ischemia also triggers activation of several potent intracellular enzymes, we have recently investigated the effects of ischemia on Protein kinase C (PKC) activation. PKC is a potent intracellular regulatory enzyme capable of affecting contractile protein sensitivity to calcium, cytosolic calcium regulation, and substrate metabolism. Using measures of PKC activity and location (cytosol vs membrane) we have demonstrated that ischemia leads to a significant increase in PKC activity and membrane affinity, similar to that seen with direct PKC agonists such as phorbol esters. We have also shown that in the presence of circulating agonists such as pro-inflammatory cytokines (interleukin-2) similar activation of PKC does occur in association with contractile disfunction, oxygen wasting (energetic inefficiency). Other PKC agonists shown to be present after open heart surgery such as endothelin, and norepinephrine have also been shown to cause PKC activation. Based on these observations, we hypothesize that ischemia followed by reperfusion, with or without circulating PKC agonists, causes PKC activation leading to the contractile dysfunction seen late after reperfusion (6-12 hours) clinically, and that PKC mediated effects on contractile proteins (troponins I and T) and calcium regulation is the mechanism for PKC effects. We plan to 1) determine the intracellular signalling cascade responsible for PKC activation during ischemia and reperfusion with or without circulating PKC agonist, 2) establish the time course and effects of reperfusion with and without inflammatory mediators on PKC expression, and 3) evaluate the effects of calcium sensitizing inotropes on post-ischemic recovery with and without exogenous PKC agonists. Based on the information obtained we will be able to develop more specific techniques for improving post-ischemic cardiac dysfunction and energetic efficiency.